Accurate genome segregation is essential for the survival and development of all organisms. Mistakes in chromosome segregation result in cellular aneuploidies that give rise to human genetic diseases such as Down syndrome and that characterize most human cancer types. A major question in cell biology is what are the cellular mechanisms that ensure high fidelity chromosome segregation to avoid genetic instability and the resulting aneuploidy. Our studies focus on the formation and function of human centromeres and kinetochores. The functions of the kinetochore are to bind microtubules, to monitor proper chromosome attachment via the mitotic checkpoint and to segregate chromosomes in anaphase. Defects in any of these processes result in chromosome segregation errors. The centromere is sole the assembly site for the mitotic kinetochore on the chromosome. Centromere function is determined by a specialized histone variant called centromere protein A (CENP-A) and mutation or loss of CENP-A causes centromere and kinetochore dysfunction. Our first objective in this proposal is to identify the mechanisms that assemble CENP-A into chromatin. We propose to do this by identifying how two of the key proteins required for CENP-A assembly, HJURP and M18BP1, are targeted to centromeres to assemble CENP-A nucleosomes during telophase and G1. Second, we propose to characterize the mechanisms by which the essential centromere protein CENP-C interacts with arrays of centromeric chromatin. Using insights from biochemical experiments, we will test models for CENP-C function in human cells in organizing and reinforcing centromere and kinetochore structure. Third, we propose to use a novel in vitro centromere and kinetochore assembly system to understand the role of chromatin structure in promoting centromere and kinetochore function. Together our aims should provide new insight into the assembly and function of vertebrate centromeres and how their activities ensure faithful chromosome segregation.